Bridging Diagnostic Gaps: Utilising HiCrome Agar and Tetrazolium Reduction Medium for the Rapid and Presumptive Identification and Speciation of Candida Species in Vulvovaginal Candidiasis in Low-Resource Environments

Background Vulvovaginal candidiasis (VVC) is a common fungal infection caused by an overgrowth of Candida species, primarily Candida albicans (C. albicans). Using HiCrome agar and tetrazolium reduction medium offers cost-effectiveness in Candida detection by eliminating the need for additional tests, reducing equipment costs compared to automated systems, and simplifying workflow with direct species identification while maintaining high specificity. They expedite detection by directly identifying Candida species based on colony colour, bypassing the multiple steps of phenotypic methods. This efficiency saves time in the laboratory, providing rapid results without the extended processing times associated with automated systems and facilitating prompt diagnosis and treatment decisions. These diagnostic tools are especially valuable in low-resource environments where a quick and accurate diagnosis of VVC is crucial for effective treatment and management of antifungal resistance. Aims and objectives This study aims to evaluate the efficacy of HiCrome agar and tetrazolium reduction medium's efficacy in speciating Candida species in VVC cases. Materials and methods A cross-sectional observational study was conducted at Saveetha Medical College and Hospitals, Chennai, India, over six months. High vaginal swabs from 126 patients suspected of VVC were collected and plated on Sabouraud dextrose agar (SDA), HiCrome Candida differential agar (Himedia, Mumbai, India), and tetrazolium reduction medium. The results were compared with those obtained from the VITEK2 compact system (bioMérieux, Marcy-l'Étoile, France). Results Of the 126 samples, 74.6% showed single yeast infections, 7.9% displayed mixed yeast infections, and 17.5% showed no growth. A total of 114 Candida isolates were identified. Both HiCrome agar and tetrazolium reduction medium accurately identified all isolates, with complete concordance with the VITEK2 compact system. The most commonly isolated species were C. albicans (55.2%), Candida tropicalis (32.4%), Candida glabrata (8.8%), and Candida parapsilosis (3.6%). Both media provided rapid and accurate presumptive identification in low-resource settings. Conclusions HiCrome agar and tetrazolium reduction medium demonstrated high sensitivity and specificity in identifying Candida species. These methods are reliable for rapid and accurate diagnosis, particularly in resource-limited settings. However, they may require supplementary tests for definitive species identification. The adoption of these diagnostic tools represents a significant advancement in clinical microbiology, improving VVC management and addressing antifungal resistance.

Vulvovaginal candidiasis (VVC), commonly referred to as a yeast infection, is a widespread fungal infection impacting the vulva and vagina.It is predominantly caused by an overgrowth of Candida species, mainly Candida albicans, a type of yeast normally present in small quantities in the vagina, mouth, digestive tract, and on the skin [1].Disruption of the microbial balance in the vagina allows Candida to multiply excessively, leading to infection.This condition affects millions of women worldwide, causing significant discomfort and disruption to daily life [2].Candida species typically coexist with protective bacteria in the vagina, maintaining an acidic environment that prevents the overgrowth of pathogens.Factors such as uncontrolled diabetes, antibiotic use, hormonal changes, immunosuppression, and diet and lifestyle choices can disrupt this delicate balance, increasing susceptibility to VVC [3].Antibiotics can reduce the population of protective bacteria, while hormonal fluctuations, immunosuppression, and poor diet or hygiene can also contribute to yeast overgrowth [4].When Candida proliferates, it adheres to vaginal epithelial cells, forming biofilms that enhance its survival and resistance to treatment.This overgrowth triggers an inflammatory response, causing the characteristic symptoms of VVC [5].Symptoms may vary in intensity and encompass strong itching and discomfort of the vulva and vaginal area, a dense, white, cottage cheese-like discharge without an unpleasant smell, inflammation and enlargement of the vulva and vaginal tissues, discomfort during sexual intercourse and urination, and a sensation of burning in the affected region [6].Diagnosing VVC involves a comprehensive approach that includes clinical assessment and laboratory tests.A typical diagnostic protocol includes a clinical examination to evaluate symptoms and perform a physical assessment of the vulva and vagina.Microscopy is used to examine a sample of vaginal discharge to detect Candida cells and pseudo hyphae indicative of a yeast infection.Vaginal cultures are also used to grow and identify Candida species, although this is not always necessary for uncomplicated cases.Additionally, vaginal pH testing, which is usually below 4.5 in VVC, helps differentiate it from conditions like bacterial vaginosis, which is characterised by elevated pH levels.Managing recurrent or complicated cases may require additional testing to identify underlying conditions or resistant Candida species [7][8][9].Treatment for VVC focuses on eradicating the infection, alleviating symptoms, and preventing recurrence.Antifungal medications are the primary treatment option and include topical antifungals such as over-the-counter and prescription creams, ointments, suppositories, and tablets containing agents like clotrimazole, miconazole, and tioconazole, which are applied directly to the affected area.Oral antifungals like fluconazole are used for severe or recurrent infections, with a single dose often being effective, though chronic cases may require longer courses [10].VVC is the most common yeast infection in humans, affecting about 75% of women at least once in their lifetime.Recurrent VVC (RVVC) occurs when a person has more than three episodes in a year and affects almost 8% of women globally.It often needs ongoing antifungal treatment with azole drugs to reduce the chance of the infection coming back [11].The persistent effectiveness of azoles and insufficient immune-mediated clearance are important contributors to the reoccurrence of the disease [12].
Identifying the specific Candida species responsible for the infection is crucial due to variations in their susceptibility to antifungal medications.For example, Candida glabrata (C.glabrata) often shows resistance to azoles, necessitating higher doses or alternative antifungal agents like echinocandins [13].Similarly, Candida krusei (C.krusei) is inherently resistant to fluconazole, requiring alternative treatments.Accurate speciation enables targeted therapy, ensuring the most effective antifungal agent is selected, minimising the risk of treatment failure.In recurrent or chronic VVC, speciation is even more important because non-albicans Candida (NAC) species, which are often more resistant to standard treatments, are frequently involved.Identifying the specific species helps tailor treatment regimens and allows for early intervention in cases where resistance to standard treatments is detected [14].Detecting mixed infections with multiple yeast species in clinical samples is crucial for effective patient treatment.The challenge of quickly identifying these mixed cultures on traditional Sabouraud's dextrose agar (SDA) has prompted the development of chromogenic media.This advancement not only helps in the rapid identification of mixed yeast infections from clinical samples but also provides results 24-48 hours earlier compared to standard identification methods.HiCrome agar and tetrazolium reduction medium are valuable tools in the laboratory for the presumptive speciation of Candida in mixed yeast infections [15].These media enable the differentiation and presumptive identification of Candida species based on their unique biochemical properties and colony morphology.HiCrome agar contains substrates that react with specific enzymes produced by different Candida species, resulting in colonies displaying distinct colours [16].Tetrazolium reduction agar differentiates Candida species based on their ability to reduce tetrazolium salt into insoluble coloured formazans resulting in colour change [17].Both media provide a relatively quick, visual, costeffective, and accurate method for identifying Candida species, aiding in appropriate treatment selection and improving patient outcomes in antifungal resistance cases.Despite some limitations, these methods are invaluable in the clinical microbiology laboratory for managing Candida-related infections.Therefore, our study aims to evaluate the effectiveness and utility of HiCrome agar and tetrazolium reduction medium in the speciation of Candida species in cases of VVC.

Study design and setting
This cross-sectional observational study was conducted at the Central Laboratory, Department of Microbiology, Saveetha Medical College and Hospitals, Chennai, India.The study was carried out over six months, from December 2023 to May 2024, after obtaining ethical clearance (258/07/2023/PG/SRB/SMCH). High vaginal swabs were collected from 126 patients suspected of VVC.

Inclusion criteria
Women diagnosed with VVC, based on clinical symptoms and the presence of vaginal discharge, were included in the study.

Exclusion criteria
Women with co-infections (e.g., bacterial vaginosis, trichomoniasis), pregnant women, and those who have used antifungal treatments within the last month were excluded from the study.
The samples were then directly plated on SDA, HiCrome Candida differential agar (Himedia, Mumbai, India), and tetrazolium reduction agar, and incubated at 37°C for 48 hours aerobically.

HiCrome Candida differential agar
The HiCrome Candida differential agar is a selective and differential chromogenic medium designed to distinguish between Candida species.Inoculate the clinical sample onto the HiCrome Candida differential agar and incubate at 37°C for 24-48 hours aerobically.Using specific enzymatic activities and various chemical substrates, the medium causes Candida colonies to display different colours, allowing for presumptive identification on the isolation plate, as referenced in Table 1.

Candida albicans
Light green

Candida parapsilosis
White to light pink

Tetrazolium reduction medium
The tetrazolium reduction medium is utilised for distinguishing between different Candida species based on the colours they produce.Various species of Candida reduce tetrazolium to varying extents.To prepare the medium, dissolve 1 gm of peptone, 4 gm of glucose, and 0.1 gm of beef extract in 100 mL of distilled water and adjust the pH to between 5.6 and 6.2.After autoclaving at 121°C for 15 minutes, cool to a temperature that can be touched by hand and add 20 mg of tetrazolium and 50 mg of neomycin.Inoculate the clinical sample onto the tetrazolium reduction medium and incubate at 37°C for 24-48 hours aerobically.After incubation, the resulting colours are observed for presumptive identification [18], as shown in Table 2.

Candida albicans Light pink
Candida tropicalis Dark, maroon-red

Candida parapsilosis
Rose pink

Candida krusei
Pink and dry Clinical samples with single yeast infections on HiCrome agar and tetrazolium reduction medium were directly identified using the VITEK2 system (bioMérieux, Marcy-l'Étoile, France) from the SDA.Samples showing mixed yeast infections with multiple Candida species on HiCrome agar and tetrazolium reduction medium were cultured again on SDA and incubated aerobically at 37°C for 24-48 hours.Pure colonies from these cultures were then identified and confirmed using the VITEK2 system.This method ensured precise identification of both single and mixed Candida infections, utilising the specific screening capability of chromogenic agars and the reliable species identification provided by the VITEK2 system.

Identification by VITEK2 system
For identification using the VITEK2 system, a pure culture of the organism was suspended in 3.0 mL of sterile saline (0.45% NaCl, pH 4.5 to 7.0) in a polystyrene test tube.Turbidity was adjusted to a McFarland 2.0 standard and measured using a DensiCHEK turbidity meter (bioMérieux, Marcy-l'Étoile, France).The culture suspension was automatically dispensed into test cards, which were sealed and placed in the VITEK2 instrument for incubation at 35.5°C for 18 hours.Optical density readings were automatically recorded every 15 minutes.The final identification results were compared against a database for accurate species identification.The evaluation included the following control strains: C. albicans ATCC 90028 and Candida tropicalis (C.tropicalis) ATCC 750.
The results from HiCrome Candida differential agar and tetrazolium reduction medium were tabulated and compared with the automated VITEK2 system to assess their effectiveness in the presumptive identification of Candida species.The data was compiled into a master chart using Microsoft Excel (Microsoft® Corp., Redmond, WA, USA), and the correlation between the media was analysed using a Chi-square test (χ² test).A p-value below 0.05 indicated statistical significance.

Results
In this study, we employed both HiCrome agar and tetrazolium reduction medium along with SDA as primary plating media for high vaginal swab samples (n = 126).Single yeast infections were observed in 74.6% of the isolates, while mixed yeast infections were identified in 7.9% of the samples.Approximately 17.5% of the samples showed no growth.A total of 114 Candida isolates were identified from the 126 samples.Using the VITEK2 compact system to identify Candida isolates, the distribution was as follows: C. albicans was the most commonly isolated species in single yeast infections (n = SDA showed creamy white to beige colonies that were smooth and pasty, measuring about 1-3 mm in diameter after 24-48 hours at 30-37°C.HiCrome Candida differential agar and tetrazolium reduction medium successfully identified all 114 Candida isolates to the species level, with complete concordance with the VITEK2 compact system results.There were no significant differences in growth rate or colony size between HiCrome agar and tetrazolium reduction medium.Both media effectively supported the growth of all isolates under standard conditions (30-37°C for 24-48 hours).The colours differed on the tetrazolium reduction medium: C. albicans appeared as light pink colonies, C. tropicalis as maroon to dark red colonies, C. parapsilosis as rose-pink colonies, and C. glabrata as bright pink colonies (Figure 2).In cases of mixed yeast infections, both HiCrome agar and tetrazolium reduction medium displayed twocoloured colonies, indicating the presence of two different Candida species, as shown in Figures 3-4.

FIGURE 3: Presumptive identification of two Candida isolates in mixed yeast infections by HiCrome agar.
Candida albicans depicted by the production of green-coloured colonies whereas Candida tropicalis is depicted by the production of steel blue colonies.

FIGURE 4: Presumptive identification of two Candida isolates in mixed yeast infections by tetrazolium reduction medium.
Candida albicans are depicted as light pink colonies, and Candida tropicalis are depicted as dark-maroon colonies.
In our study comparing the performance of HiCrome agar, tetrazolium reduction medium, and the VITEK2 compact system for the identification and speciation of Candida isolates, all three methods demonstrated complete concordance in identifying the four most common Candida species: C. albicans, C. tropicalis, C. parapsilosis, and C. glabrata.Each method accurately identified 63 isolates of C. albicans, 37 isolates of C. tropicalis, four isolates of C. parapsilosis, and 10 isolates of C. glabrata, totalling 114 isolates across all species.Statistical analysis showed no significant differences (p = 1.00) among the three methods, highlighting their comparable performance in clinical microbiology settings.These findings support the reliability and utility of both HiCrome agars and tetrazolium reduction medium as effective tools for the rapid and accurate presumptive identification of Candida species, essential for guiding appropriate antifungal therapy in cases of VVC and other Candida-related infections.The comparison of the two chromogenic media with the VITEK2 system was evaluated and tabulated, as shown in  The data are presented as numbers (N).The p-value is calculated using the Chi-square test, with significance considered when the p-value is less than 0.05.In this case, the p-value is 1.00, which is considered not significant.

Discussion
C. albicans remains the predominant species isolated in cases of VVC, consistent with several studies where C. albicans accounted for a significant majority of isolates [19][20][21].Notably, our study also highlights a substantial prevalence of mixed yeast infections (17%), which is higher than in three other studies, which had prevalence rates of 13%, 5%, and 0.12% [22][23][24].Traditionally, laboratories faced challenges in accurately identifying NAC species, relying primarily on the germ tube test for initial identification without subsequent species-level testing or antifungal susceptibility profiling.This approach, prevalent in resourcelimited settings, often leads to suboptimal diagnosis and treatment decisions, potentially contributing to the emergence of antifungal resistance [25,26].In our study, we evaluated the efficacy of HiCrome agar and tetrazolium reduction medium in identifying Candida species based on colony morphology and colour differentiation.Both methods demonstrated high sensitivity and specificity, accurately identifying all 114 Candida isolates, including mixed infections.This aligns with previous studies by Nadeem et al. [25] and Pravin Charles et al. [27] affirming the reliability of these mediums in clinical settings.While the literature on tetrazolium reduction medium for Candida species identification is limited, Giri and Kindo [18] demonstrated its reliability in speciation.Similarly, Denny and Partridge [17] found that the tetrazolium reduction medium is a rapid, fairly accurate, and straightforward method for differentiating Candida species from other yeasts in mixed yeast infections, especially in vaginal samples.Our study confirms these findings, as tetrazolium reduction medium successfully speciated all Candida isolates, including those from both single and mixed yeast infections.
Chromogenic agars emerged as a pivotal tool in our research, enabling the early identification of Candida species within mixed cultures based solely on colony colour.This capability is crucial as conventional methods often fail to detect mixed infections accurately.The research conducted by Willinger and Manafi emphasised the utility of chromogenic agars for isolating and presumptively identifying yeasts, particularly in detecting mixed cultures in clinical samples.They reported high specificity rates: 98.8% for C. albicans, 99.8% for C. tropicalis, and 100% for C. krusei.Our study supports these findings, with our chromogenic media successfully identifying all tested Candida species presumptively.This underscores the effectiveness of chromogenic agars in identifying mixed infections, thereby improving treatment approaches and preventing drug resistance [22].Comparison with the VITEK2 compact system showed no statistical difference in Candida identification and speciation between chromogenic mediums and this automated system.This suggests that chromogenic agars can serve as a reliable alternative for presumptive identification and speciation in mixed yeast infections of VVC, particularly in settings where rapid diagnosis is imperative.In clinical practice, swiftly and accurately identifying Candida species is crucial for guiding appropriate antifungal therapy.Advanced diagnostic tools like chromogenic agars and tetrazolium reduction medium represent a significant advancement, particularly in regions with limited resources and expertise.Future research to enhance the diagnostic process for identifying Candida in low-resource settings using HiCrome agar and tetrazolium reduction medium could focus on several key areas.Incorporating novel biomarkers or genetic markers could lead to more accurate and rapid identification of Candida species, reducing false negatives and positives.Optimising the nutrient and chromogenic substrate composition can improve growth and colour differentiation, making it easier to distinguish closely related species.Using multiple chromogens targeting different enzymatic activities and adding selective agents to inhibit nontarget organisms can further enhance specificity and reduce misidentification, improving diagnostic accuracy.These methods streamline diagnosis and facilitate targeted treatment approaches, thereby mitigating the risk of antifungal resistance.Additionally, tetrazolium reduction medium may inaccurately differentiate species, increasing the risk of false results, particularly in cases involving non-Candida yeasts in mucosal or skin scrapings [17].

Limitations of our study
Non-Candida yeasts tend to grow poorly on tetrazolium reduction medium, so it is necessary to use phenotypic and automated methods, especially emerging ones, for identification and confirmation.Factors such as pH, temperature, and medium composition can influence the reduction process, potentially leading to inconsistent results.Future research to improve Candida species identification should focus on developing chromogenic and tetrazolium reduction media with enhanced specificity and sensitivity.Expanding reference databases with comprehensive genetic and phenotypic profiles of Candida species, including rare strains, will improve accuracy.

Conclusions
Our research underscores the importance of utilising advanced diagnostic tools such as chromogenic agars and tetrazolium reduction medium to accurately identify Candida species linked to VVC.These methods have demonstrated exceptional sensitivity and specificity in identifying C. albicans, C. tropicalis, C. parapsilosis, and C. glabrata, essential for guiding effective antifungal therapy and addressing antimicrobial resistance in mixed yeast infections.However, it's important to recognise the limitations of chromogenic agars, as they may not always provide definitive species identification, necessitating additional testing for confirmation.Despite these challenges, adopting these diagnostic tools represents a notable advancement in clinical microbiology, particularly valuable in resource-limited settings.Future research should continue to explore and refine these diagnostic modalities to further enhance their efficacy and accessibility in clinical practice.

FIGURE 1 :
FIGURE 1: Presumptive identification of Candida isolates based on colour production by HiCrome agar.A) Green-coloured colonies indicate Candida albicans; B) White to cream-coloured colonies indicate Candida glabrata; C) Steel blue colonies indicate Candida tropicalis; D) Pink-coloured colonies indicate Candida parapsilosis

FIGURE 2 :
FIGURE 2: Presumptive identification of Candida isolates by tetrazolium reduction medium.A) Candida albicans: light pink colonies; B) Candida parapsilosis: rose-pink colonies; C) Candida tropicalis: maroon to dark red colonies; D) Candida glabrata: bright pink colonies

TABLE 2 : Identification of Candida species based on colour production by TRM.
TRM: Tetrazolium reduction medium

TABLE 3 : The total number of isolates of Candida identified by the VITEK2 compact system and two media.
HiCrome agar aids in distinguishing Candida species by colony colour and morphology but may not always provide definitive species identification.Supplementary biochemical or molecular tests are often required for accuracy.Interpretation of colony colours and morphology is subjective, demanding experienced personnel for precision.Variations in colony colour, like different shades of green between closely related species such as C. albicans and Candida dubliniensis (C.dubliniensis), can cause confusion.However, relying solely on colour for identification is insufficient, especially for pathogens like Candida auris (C.auris), Candida kefyr (C.kefyr), and Candida famata (C.famata), which lack specific colour markers.